硒化乌拉尔甘草多糖和乌拉尔甘草多糖对单核细胞增生李斯特菌抗菌活性的研究

Study on the antibacterial activity of selenizing Glycyrrhiza uralensis polysaccharide and Glycyrrhiza uralensis polysaccharide against Listeria monocytogenes

为了比较硒化乌拉尔甘草多糖(selenizing Glycyrrhiza uralensis polysaccharide,SeGUP)和乌拉尔甘草多糖(Glycymhiza uralensis polyacchiade,GUP)对单核细胞增生李斯特菌(Listeria monocytogene,Lm)体内、体外抗菌活性,试验采用药敏纸片方法分别测定Lm对480 mg/mL、240 mg/mL、120 mg/mL GUP和SeGUP的敏感程度;采用微量肉汤稀释法测定SeGUP和GUP对Lm的最小抑菌浓度(MIC)和最小杀菌浓度(MBC);设置SeGUP和GUP高剂量(240 mg/mL)组、中剂量(120 mg/mL)组、低剂量(60 mg/mL)组,各组分别与Lm混合培养24 h,每2 h取样测定OD600值,观察SeGUP、GUP对Lm生长速率的影响;检测各组4小时时的培养上清液中的碱性磷酸酶(AKP)、β-半乳糖苷酶活性及蛋白质总含量;用结晶紫染色法检测SeGUP和GUP各剂量组对Lm生物被膜形成能力的影响;分别按体重灌胃小鼠SeGUP、GUP 300 mg/kg、150 mg/kg、75 mg/kg测定其对感染Lm小鼠的保护效果。结果表明:480 mg/mL SeGUP对Lm高度敏感,抑菌圈直径为15.0 mm;480 mg/mL GUP中度敏感,抑菌圈直径为13.0 mm。SeGUP和GUP对Lm的MIC分别为120 mg/mL、240 mg/mL,MBC分别为240 mg/mL、480 mg/mL。各剂量SeGUP和GUP均能抑制Lm的生长,其中240 mg/mL SeGUP抑制效果最好;与空白对照组相比,SeGUP和GUP各剂量组的AKP、β-半乳糖苷酶活性及蛋白质总含量均显著升高(P<0.05),各剂量组均能显著抑制生物被膜形成(P<0.05);与模型组相比,300 mg/kg、150 mg/kg、75 mg/kg SeGUP组和300 mg/kg、150 mg/kg GUP组小鼠死亡率均显著降低(P<0.05),75 mg/kg GUP组小鼠死亡率差异不显著(P>0.05);各SeGUP组小鼠死亡率低于相同浓度的GUP组。说明SeGUP、GUP对Lm生长有明显的抑制作用,相同浓度时SeGUP的效果更好,并且SeGUP浓度为240mg/mL时体外抗菌效果最好,按体重灌胃剂量为300 mg/kg时对小鼠的保护力最强。

In order to study the in vivo and in vitro antibacterial activity of selenizing Glycyrrhiza uralensis polysaccharide(SeGUP)and Glycyrrhiza uralensis polysaccharide(GUP)on Listeria monocytogenes(Lm),in this experiment,the sensitivity of 480 mg/mL,240 mg/mL and 120 mg/mL of GUP and SeGUP to Lm was determined by the antimicrobial paper method.The minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of SeGUP and GUP on Lm were determined by microbroth dilution method.SeGUP and GUP high-dose(240 mg/mL)groups,medium-dose(120 mg/mL)groups,and low-dose(60 mg/mL)groups were set up.Each group was incubated with Lm for 24 h,and OD600 values were sampled every 2 h to observe the effects of SeGUP and GUP on Lm growth rate.The alkaline phosphatase(AKP),β-galactosidase activity and total protein content in the culture supernatant of each group at 4 h were detected by ELISA method.The effects of SeGUP and GUP dose groups on the formation capacity of Lm biofilm were detected by crystal violet staining method.The protective effects of SeGUP and GUP 300 mg/kg,150 mg/kg and 75 mg/kg by gavage respectively in mice infected with Lm were determined according to body weight of mice.The results showed that Lm was highly sensitive to 480 mg/mL SeGUP,with an inhibitory diameter of 15.0 mm,moderately sensitive to 480 mg/mL GUP with an inhibitory diameter of 13.0 mm.The MICs of SeGUP and GUP for Lm were 120 mg/mL and 240 mg/mL,respectively,and the MBCs were 240 mg/mL and 480 mg/mL,respectively.Both doses of SeGUP and GUP could inhibit the growth of Lm,and 240 mg/mL SeGUP had the best inhibition effect.Compared with the blank control group,the AKP,β-galactosidase activity and total protein content of SeGUP and GUP groups were significantly increased(P<0.05),and biofilm formation was significantly inhibited in all dose groups(P<0.05).Compared with the model group,the mortality rates of 300 mg/kg,150 mg/kg,75 mg/kg SeGUP groups and 300 mg/kg and 150 mg/kg GUP groups were significantly reduced(P<0.05),while the mortality difference in GUP 75 mg/kg group was not significant(P>0.05),and the mortality rate of mice in each SeGUP group was lower than that in the GUP group with the same concentration.The results suggested that SeGUP and GUP had obvious inhibitory effect on Lm growth,and the effect of SeGUP was better at the same concentration,and the antibacterial effect in vitro was the best when the SeGUP concentration was 240 mg/mL,and the strongest protection for mice was at the dose of 300 mg/kg according to body weight by gavage.